Bradyrhizobium japonicum glnB, a Putative Nitrogen-Regulatory

Vol. 171, No. 10

JOURNAL OF BACTERIOLOGY, OCt. 1989, p. 5638-5645

0021-9193/89/105638-08$02.00/0 Copyright © 1989, American Society for Microbiology

Bradyrhizobium japonicum glnB, a Putative Nitrogen-Regulatory Gene, Is Regulated by NtrC at Tandem Promoterst GREGORY B. MARTIN,'12t* MICHAEL F. THOMASHOW,'34 AND BARRY K. CHELM" 3§ MSUIDOE Plant Research Laboratory,' Genetics Program,2 Department of Microbiology,3 and Department of Crop and Soil Sciences,4 Michigan State University, East Lansing, Michigan 48824-1312 Received 27 April 1989/Accepted 20 July 1989 The glnB gene from Bradyrhizobium japonicum, the endosymbiont of soybeans (Glycine max), was isolated and sequenced, and its expression was examined under various culture conditions and in soybean nodules. The B. japonicum glnB gene encodes a 12,237-dalton polypeptide that is highly homologous to the glnB gene products from KlebsieUa pneumoniae and Escherichia coli. The gene is located directly upstream from ginA (encoding glutamine synthetase), a linkage not observed in enteric bacteria. The glnB gene from B. japonicum is expressed from tandem promoters, which are differentially regulated in response to the nitrogen status of the medium. Expression from the downstream promoter involves the B. japonicum ntrC gene product (NtrC) in both free-living and symbiotic cells. Thus, glnB, a putative nitrogen-regulatory gene in B. japonwum, is itself Ntr regulated, and NtrC is active in B. japonicum cells in their symbiotic state.

Bradyrhizobium japonicum exists as a free-living organism, growing at the expense of soil nitrogen, or as a symbiont, reducing dinitrogen to ammonia for itself and its soybean host. Free-living B. japonicum cells assimilate ammonia primarily by the coordinate activities of glutamine synthetase (GS) and glutamate synthase (12, 56). However, during the development of bacteroids, the symbiotic form of these bacteria, GS activity decreases in concert with the derepression of nitrogen fixation activity (12, 56). The regulation of nitrogen assimilation pathways is therefore an integral part of the bacterial developmental process and the symbiotic interaction. In the family Enterobacteriaceae many genes involved in nitrogen assimilation, including glnA, encoding GS, are regulated by the Ntr system (36, 40, and references cited below). This regulation involves both transcriptional and posttranslational control exerted by the products of the ntrA, ntrB, ntrC, glnB, glnD, and glnE genes (NtrA, NtrB, NtrC, GlnB, GlnD, and GinE, respectively [8, 10, 14, 23, 28, 31, 32, 35]). NtrB and NtrC are bifunctional regulatory proteins that act in concert to either repress or activate transcription (35, 43, 47). The functional state of NtrB is modulated by GlnB and GlnD in response to the ratio of intracellular glutamine to 2-ketoglutarate (14). Depending on its state, NtrB is able to switch NtrC between the activating phosphorylated form and the repressing dephosphorylated form (33, 41). NtrA is an alternative sigma factor that confers specificity on core RNA polymerase for NtrA-specific promoter sequences (28, 31). GlnB (PI, protein) plays a central role in coordinating the response of the Ntr system to combined nitrogen (13, 36, 37, 53). It is a tetrameric protein with a subunit molecular mass of 12,387 daltons (29, 53) and exists in two interconvertible forms (13). Under nitrogen deprivation conditions, GlnB is *

Corresponding author.

t Michigan Agricultural Experiment Station article 13059. t Present address: Department of Plant Breeding and Biometry, Cornell University, Ithaca, NY 14853. § Deceased 2 September 1987.

uridylylated at a tyrosyl residue by GlnD, a uridylytransferase (14, 53). Under conditions of nitrogen excess GlnB is deuridylylated by GlnD (14). In the uridylylated form, GlnB stimulates the adenylyltransferase activity of GlnE, which mediates the deadenylylation (activation) of GS (4, 32). In the deuridylylated form, GlnB acts in concert with GlnE to adenylylate (inactivate) GS (32). The deuridylylated GInB also interacts with NtrB to dephosphorylate (inactivate) NtrC under conditions of nitrogen excess (23, 36). Thus, GlnB in the enteric bacteria is centrally involved in transmitting the nitrogen status of the cell to the GS-modifying enzyme, GlnE, and the NtrC-modifying enzyme, NtrB. Expression of nitrogen assimilation capacity in B. japonicum is coordinated by separate regulatory networks that respond to nitrogen or oxygen limitation (2, 17, 39). The network responsive to nitrogen status shares many features with Ntr control in members of the Enterobacteriaceae (26, 36), and genes that have functional roles similar to those of the ntrC and ntrA genes of enteric bacteria have been characterized from several rhizobial species (39, 44, 50, 51, 54). The regulatory network responsive to oxygen limitation is activated in symbiotic cells and probably relies on the positive regulator NifA to activate the genes controlled by this system (39). Most members of the family Rhizobiaceae contain two GS enzymes, GSI and GSII, encoded by glnA and glnII, respectively (15, 16, 20, 24). In B. japonicum, GSI is subject to adenylylation in response to ammonia, whereas GSII is not modified in this way (19, 24). Expression of glnII is controlled by both the nitrogen and oxygen regulatory networks, whereas glnA is constitutively expressed (39). The adenylylation of GSI and the presence of NtrC suggest that a functional analog of the glnB gene of enteric bacteria is present in B. japonicum. It was reported (18) that a region upstream from the Rhizobium leguminosarum glnA gene encodes a protein, part of which is highly homologous to a published 10-residue peptide sequence from the Escherichia coli glnB gene product (49). Similar homologies were noted in published partial sequences upstream from the glnA 5638

REGULATION OF B. JAPONICUM glnB GENE

VOL. 171, 1989

rrB

P2

TABLE 1. Bacterial strains, plasmids, and phage Strain, plasmid, or phage

Source or

Description

x

St

Plasmids or phage M13glnA

M13glnB

A6IG pBJ47

pBJ299

pA

S

B X

I

reference

K\ \\\

gInB

B. japonicum

strains BJl1Od BJ27147 BJ3028

5639

g

\ \ \ \ "

'

__

ginA

100bp

Wild type ntrC::nptII, null mutant; Kmr ntrC::nptII, truncated NtrC; Kmr

25 39 39

391-bp SalI fragment from B. japonicum glnA promoter (16) region cloned into M13mp18 (57) 1,000-bp EcoRI-SstI fragment from B. japonicum glnB region cloned into M13mp19 (57) Recombinant phage carrying entire B. japonicum glnB and glnA genes in XBF101 (38) Apr; 500-bp EcoRI-BamHI fragment containing entire B. japonicum glnB gene cloned into pBR322 (38) Kmr Apr; 2.3-kbp XhoI fragment containing the B. japonicum glnB gene and partial glnA gene from X61G cloned into pKC7 (46)

2

I

,--